Inhibition of xanthine oxidase alleviated pancreatic necrosis via HIF-1α-regulated LDHA and NLRP3 signaling pathway in acute pancreatitis.

Acute pancreatitis (AP) is a potentially fatal condition with no targeted treatment options. Although inhibiting xanthine oxidase (XO) in the treatment of AP has been studied in several experimental models and clinical trials, whether XO is a target of AP and what its the main mechanism of action is remains unclear. Here, we aimed to re-evaluate whether XO is a target aggravating AP other than merely generating reactive oxygen species that trigger AP. We first revealed that XO expression and enzyme activity were significantly elevated in the serum and pancreas of necrotizing AP models. We also found that allopurinol and febuxostat, as purine-like and non-purine XO inhibitors, respectively, exhibited protective effects against pancreatic acinar cell death in vitro and pancreatic damage in vivo at different doses and treatment time points. Moreover, we observed that conditional Xdh overexpression aggravated pancreatic necrosis and severity. Further mechanism analysis showed that XO inhibition restored the hypoxia-inducible factor 1-alpha (HIF-1α)-regulated lactate dehydrogenase A (LDHA) and NOD-like receptor family pyrin domain containing 3 (NLRP3) signaling pathways and reduced the enrichment of 13C6-glucose to 13C3-lactate. Lastly, we observed that clinical circulatory XO activity was significantly elevated in severe cases and correlated with C-reactive protein levels, while pancreatic XO and urate were also increased in severe AP patients. These results together indicated that proper inhibition of XO might be a promising therapeutic strategy for alleviating pancreatic necrosis and preventing progression of severe AP by downregulating HIF-1α-mediated LDHA and NLRP3 signaling pathways.

Efficacy and safety of the urate-lowering agent febuxostat in chronic heart failure patients with hyperuricemia: results from the LEAF-CHF study.

Hyperuricemia is an independent predictor of mortality in patients with chronic heart failure (CHF). To determine whether febuxostat, a urate-lowering agent, may improve clinical outcomes in CHF patients, we conducted a multicenter, prospective, randomized, open-label, blinded endpoint study with a treatment period of 24 weeks. We randomly assigned Japanese outpatients diagnosed with both CHF with reduced left ventricular ejection fraction (LVEF < 40%) and asymptomatic hyperuricemia (serum uric acid [UA] levels > 7.0 mg/dl and < 10.0 mg/dl) to either a febuxostat group (n = 51) or a control group (n = 50). The primary efficacy endpoint was the change in log-transformed plasma B-type natriuretic peptide (BNP) levels from baseline to week 24 (or at discontinuation). The secondary efficacy endpoints were the changes in LV systolic or diastolic function evaluated by echocardiography, New York Heart Association (NYHA) class, hemoglobin, and estimated glomerular filtration rate from baseline to week 24, and the change in log-transformed plasma BNP levels or serum UA levels from baseline to weeks 4, 8, 12, 16 and 20 (BNP) or weeks 4, 8, 12, 16, 20 and 24 (serum UA). The primary safety endpoints were occurrence of all-cause death or major cardiovascular events. The mean age of participants was 70 years; 14% were female. The febuxostat group and the control group did not differ with respect to the primary efficacy endpoint (p = 0.13), although the decrease in log-transformed plasma BNP levels from baseline to each of weeks 4, 8, 12, 16 and 20 was greater in the febuxostat group. There were no significant differences between the two groups in the primary safety endpoints or the secondary efficacy endpoints except reduced serum UA levels in the febuxostat group. Febuxostat did not reduce plasma BNP levels at week 24 in patients with CHF, but it appeared safe with no increase in major cardiovascular events and all-cause or cardiovascular mortality.

Quercetin as the primary xanthine oxidase inhibitor compound in Maclura tricuspidata leaf.

Maclura tricuspidata (MT) leaf demonstrated various health benefits, notably the inhibition of xanthine oxidase (XOD) activity, which is crucial in the management of hyperuricaemia and many diseases related to oxidative stress. This study aimed to identify the primary compound responsible for this inhibitory effect. Through a systematic investigation, MT leaf extracts were subjected to solvent-solvent partitioning using ethyl acetate, n-hexane, n-butanol, and dichloromethane. Further purification involved adsorption and desorption using Amberlite XAD-2 resin, followed by column chromatography on Silica Gel and Sephadex LH-20. The purified compounds were analysed using UPLC-QTOF-MS coupled with NMR spectroscopy. Our findings identified quercetin, a phenolic compound, as the most significant inhibitor of XOD activity in MT leaf, with an IC50 value of 212.92 µg/ml. This is the first report of purifying and identifying a single compound responsible for XOD inhibition in MT.

Rhabdomyolysis Associated With Febuxostat in a Non-Chronic Kidney Disease (CKD) Patient: Diagnostic and Management Challenges.

Rhabdomyolysis is a critical medical condition characterized by the rapid breakdown of muscle tissue, releasing substances such as myoglobin and creatine kinase into the bloodstream, potentially leading to acute kidney injury. The etiology includes trauma, exertion, genetic factors, infections, and adverse drug reactions. Febuxostat, a non-purine selective xanthine oxidase inhibitor used to manage hyperuricemia in gout patients, is typically well-tolerated but has been associated with rare instances of severe adverse reactions like rhabdomyolysis. This case report describes a case of a 73-year-old male who developed rhabdomyolysis shortly after initiating febuxostat for gout management. He presented with significant lower back pain and progressive leg weakness, initially suspected to be an exacerbation of his degenerative disk disease. Laboratory findings revealed alarmingly elevated creatine phosphokinase levels, and diagnostics excluded other potential etiologies. The patient's condition significantly improved following the cessation of febuxostat and initiation of supportive care, including high-dose intravenous corticosteroids and hydration. This case underscores the need for vigilance in monitoring for rhabdomyolysis in patients starting febuxostat, especially when presenting with unexplained muscle weakness or pain.

A potential therapeutic agent for the treatment of hyperuricemia and gout: 3,4-Dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazide.

Uric acid, the metabolic product of purines, relies on xanthine oxidase (XOD) for production. XOD is a target for the development of drugs for hyperuricemia (HUA) and gout. Currently, treatment options remain limited for gout patients. 3, 4-Dihydroxy-5-nitrobenzaldehyde (DHNB) is a derivative of the natural product protocatechualdehyde with good biological activity. In this work, we identify a DHNB thiosemicarbazide class of compounds that targets XOD. 3,4-Dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazone can effectively inhibit XOD activity (IC50 value: 0.0437 µM) and exhibits a mixed inhibitory effect. In a mouse model of acute hyperuricemia, a moderate dose (10 mg/kg.w) of 3,4-dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazide effectively controlled the serum uric acid content and significantly inhibited serum XOD activity. In addition, 3,4-Dihydroxy-5-nitrobenzaldehyde phenylthiosemicarbazide showed favorable safety profiles, and mice treated with the target compound did not show any symptoms of general toxicity following a single dose of 500 mg/kg. In the allopurinol group, 50 % of the mice died. These results provide a structural framework and mechanism of XOD inhibition that may facilitate the design of hyperuricemia and gout treatments.

Allopurinol versus Febuxostat: A New Approach for the Management of Hepatic Steatosis in Metabolic Dysfunction-Associated Steatotic Liver Disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) includes patients with hepatic steatosis and at least one of five cardiometabolic risk factors. Xanthine oxidase (XO) represents a treatment target for MASLD. We aimed to evaluate the effect of two xanthine oxidase inhibitors, allopurinol and febuxostat, plus lifestyle modifications compared to lifestyle modifications alone on improving steatosis. Ninety MASLD patients were assigned to one of three groups for three months. Patients with hyperuricemia were given either allopurinol 100 mg or febuxostat 40 mg daily, along with lifestyle modifications. The third control group was only given lifestyle modifications, excluding all patients with hyperuricemia due to ethical concerns. The primary outcome was to measure the change in the controlled attenuation parameter (CAP) score as an indicator of steatosis from baseline after three months. The secondary outcome was to measure the change in serum uric acid (SUA) three months from baseline. The study found that the CAP score decreased significantly in the allopurinol group (p = 0.009), but the decline in the febuxostat or lifestyle groups was non-significant (p = 0.189 and 0.054, respectively). The SUA levels were significantly reduced in both the allopurinol and febuxostat groups (p < 0.001), with no statistical difference between the two groups (p = 0.496).

The Stiff Joint: Comparative Evaluation of Monotherapy and Combination Therapy With Urate Lowering Agents in Managing Acute Gout.

Gout, an extremely painful form of arthritis, is triggered by the innate immune system's response to the accumulation of monosodium urate crystals in specific joints and surrounding tissues. This condition is characterized by recurring episodes of excruciating arthritis flares, interspersed with periods of disease quiescence. Over time, gout can result in disability, tophi formation, and severe pain. The treatment of gout is centered around two main objectives: alleviating inflammation and pain during acute gout attacks and long-term management to reduce serum urate levels and mitigate the risk of future attacks. Addressing inflammation and pain during acute attacks is often complicated by various factors, including underlying health conditions commonly associated with gout, such as hypertension, chronic kidney disease, cardiovascular disease, and diabetes mellitus. Moreover, gout patients are frequently older and have multiple coexisting health issues, necessitating complex medication regimens. Given the rising prevalence of gout and its associated comorbidities, there's a growing demand for improved treatment options. While existing treatments effectively manage gout in some patients, a significant portion, particularly those with comorbidities, face contraindications to these treatments and require alternative approaches. Innovative medications are required to enhance gout treatment, especially for individuals with concurrent health conditions. These considerations underscore the importance of reviewing both monotherapy and combination therapy approaches for acute gout treatment.

New Guaiane-Type Sesquiterpene and Norsesquiterpene from Alisma plantago-aquatica and Their Xanthine Oxidase Inhibitory Activity.

A new sesquiterpene (1) and a new norsesquiterpene (2) belonging guaiane-type skeleton together with six known compounds (3-8) were isolated from the rhizomes of Alisma plantago-aquatica. Their structures were determined by HR-ESI-MS, 1D and 2D NMR spectroscopic methods. Absolute configurations of new compounds were established by experimental and TD-DFT computational ECD spectra. Compounds 1-8 exhibited xanthine oxidase inhibitory activity with their IC50 values in range of 9.4-66.7 µM. The sesquiterpenoids 1-5 displayed the inhibitory activity and hence they could be potential xanthine oxidase inhibitors from A. plantago-aquatica.

Therapeutic voyage of synthetic and natural xanthine oxidase inhibitors.

Xanthine oxidase (XO) inhibitors are commonly used to treat gout, nephropathy, and renal stone diseases related to hyperuricemia. However, recent research has shown that these inhibitors may also have potential benefits in preventing vascular diseases, including those affecting the cerebrovasculature. This is due to emerging evidence suggesting that serum uric acid is involved in the growth of cardiovascular disease, and XO inhibition can reduce oxidative stress in the vasculature. There is a great interest in the development of new XO inhibitors for the treatment of hyperuricemia and gout. The present review discusses the many synthetic and natural XO inhibitors that have been developed which are found to have greater potency.

Quantitative structure-activity relationship study of amide derivatives as xanthine oxidase inhibitors using machine learning.

The target of the study is to predict the inhibitory effect of amide derivatives on xanthine oxidase (XO) by building several models, which are based on the theory of the quantitative structure-activity relationship (QSAR). The heuristic method (HM) was used to linearly select descriptors and build a linear model. XGBoost was used to non-linearly select descriptors, and radial basis kernel function support vector regression (RBF SVR), polynomial kernel function SVR (poly SVR), linear kernel function SVR (linear SVR), mix-kernel function SVR (MIX SVR), and random forest (RF) were adopted to establish non-linear models, in which the MIX-SVR method gives the best result. The kernel function of MIX SVR has strong abilities of learning and generalization of established models simultaneously, which is because it is a combination of the linear kernel function, the radial basis kernel function, and the polynomial kernel function. In order to test the robustness of the models, leave-one-out cross validation (LOOCV) was adopted. In a training set, R2 = 0.97 and RMSE = 0.01; in a test set, R2 = 0.95, RMSE = 0.01, and Rcv2 = 0.96. This result is in line with the experimental expectations, which indicate that the MIX-SVR modeling approach has good applications in the study of amide derivatives.

Association between urate-lowering therapy and cardiovascular events in patients with asymptomatic hyperuricemia.

INTRODUCTION/OBJECTIVES: To investigate the role of urate-lowering therapy (ULT) in the prevention of cardiovascular disease (CVD) in patients with asymptomatic hyperuricemia using the Japanese healthcare record database. METHODS: This retrospective cohort study used data from the JMDC Claims Database, which includes records of medical check-ups and Japanese health insurance claims. Subjects aged at least 18 years with a serum uric acid (sUA) level ≥ 7.0 mg/dL and at least one medical check-up from January 2007 to August 2021 were included in this study. The exposure was any ULT prescription, and the primary outcome included composite CVD outcomes, including coronary artery disease, stroke, and atrial fibrillation. Analysis was performed with a new-user design and overlap weighting to balance the baseline characteristics of the subjects. Cox proportional hazards models were used to investigate the association between ULT and the development of CVD. RESULTS: In total, 152,166 patients were included in the main analysis before overlap weighting in this retrospective cohort study. The number of subjects in the ULT group was 5,270, and there were 146,896 subjects in the control group. Composite CVD outcomes were observed in a total of 7,703 patients. The risk of developing composite CVD outcomes was not different between the ULT group and the control group (HR: 1.01, 95% CI: 0.89 to 1.13). CONCLUSIONS: ULT for patients with asymptomatic hyperuricemia did not prevent the development of CVD based on the Japanese claims database. Key points • Among subjects with asymptomatic hyperuricemia, ULT was not associated with a lower risk of CVD • There was no appropriate cutoff for initiating ULT in patients with asymptomatic hyperuricemia • There was no appropriate cutoff as the therapeutic goal of ULT in patients with asymptomatic hyperuricemia.

A novel multi-hyphenated approach to screen and character the xanthine oxidase inhibitors from saffron floral bio-residues.

Recently, the incidence of hyperuricemia increased with patient rejuvenation, searching for new xanthine oxidase (XOD) inhibitors from natural products becomes important. In our previous work, a flavonoid extract of saffron floral bio-residues (SFB) was found to alleviate hyperuricemia via inhibiting XOD. In this study, an integrated approach combining two-dimensional liquid chromatography, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) was developed to online screen and character the potential XOD inhibitors from SFB. The two-dimensional liquid chromatography consisted of affinity chromatography and reverse phase chromatography (2D-AR), in which an XOD column, an inactive XOD column, and a control column were used in the first dimensional liquid chromatography to avoid phenomena of "false positive" and "missing screen of compounds with weak affinity to XOD" that often occur in the screening process, and a C18 column was used in the second dimensional liquid chromatography to separate the mixed XOD binders. Four flavonoid glycosides, i.e., quercetin-3-O-sophoroside (QS), kaempferol-3-O-sophoroside (KS), kaempferol-3-O-rutinoside (KR), and kaempferol-3-O-glucoside (KG), were thus successfully screened and identified from SFB extract by the 2D-AR method. The affinity of QS, KS, KR, KG, kaempferol (aglycone of KS, KR and KG), and quercetin (aglycone of QS) binding to XOD was investigated using SPR method, with KD ranged from 4.8 µM to 47.6 µM. The inhibitor constant (KI) of KS, KR, KG, quercetin and kaempferol were 4.92 mM, 1.11 mM, 0.294 mM, 4.93 µM and 3.27 µM, respectively, determined using ITC method. Finally, the anti-XOD activities of KS, the most abundant flavonoid in SFB extract, and kaempferol in hyperuricemia mice were verified, which suggested that the multi-hyphenated approach established herein can be applied for screen and character the XOD inhibitors in natural products.

Investigating the Vital Role of the Identified Abietic Acid from Helianthus annuus L. Calathide Extract against Hyperuricemia via Human Embryonic Kidney 293T Cell Model.

To explore the anti-hyperuricemia components in sunflower (Helianthus annuus L.) calathide extract (SCE), we identified abietic acid (AA) via liquid chromatography-mass spectrometry and found an excellent inhibitor of xanthine oxidase (IC50 = 10.60 µM, Ki = 193.65 nM) without cytotoxicity. Based on the transcriptomics analysis of the human embryonic kidney 293T cell model established using 1 mM uric acid, we evaluated that AA showed opposite modulation of purine metabolism to the UA group and markedly suppressed the intensity of purine nucleoside phosphorylase, ribose phosphate pyrophosphokinase 2, and ribose 5-phosphate isomerase A. Molecular docking also reveals the inhibition of purine nucleoside phosphorylase and ribose phosphate pyrophosphokinase 1. The SCE exhibits similar regulation of these genes, so we conclude that AA was a promising component in SCE against hyperuricemia. This present study provided a novel cell model for screening anti-hyperuricemia natural drugs in vitro and illustrated that AA, a natural diterpenoid, is a potential inhibitor of purine biosynthesis or metabolism.

Studies on the interaction mechanism between xanthine oxidase and osmundacetone: Molecular docking, multi-spectroscopy and dynamical simulation.

Xanthine oxidase (XO) is a key enzyme in uric acid production, and its molybdopterin (Mo-Pt) domain is an important catalytic center when xanthine and hypoxanthine are oxidated. It is found that the extract of Inonotus obliquus has an inhibitory effect on XO. In this study, five key chemical compounds were initially identified using liquid chromatography-mass spectrometry (LC-MS), and two compounds, osmundacetone ((3E)-4-(3,4-dihydroxyphenyl)-3-buten-2-one) and protocatechuic aldehyde (3,4-dihydroxybenzaldehyde), were screened as the XO inhibitors by ultrafiltration technology. Osmundacetone bound XO strongly and competitively inhibited XO with a half-maximal inhibitory concentration of 129.08 ± 1.71 µM, and its inhibition mechanism, was investigated. Osmundacetone and XO via static quenching and spontaneously bound with XO with high affinity, primarily via hydrophobic interactions and hydrogen bonds. Molecular docking studies showed that osmundacetone was inserted into the Mo-Pt center and interacted with hydrophobic residues of Phe911, Gly913, Phe914, Ser1008, Phe1009, Thr1010, Val1011, and Ala1079 of XO. In summary, these findings suggest that provide theoretical basis for the research and development of XO inhibitors from Inonotus obliquus.

Delayed hypersensitivity reaction to allopurinol: A case report.

Allopurinol is the well-known first-line treatment option for symptomatic hyperuricaemia and gout. It is cost-effective particularly for the management of chronic gout. The common early side effects of allopurinol are skin rashes, diarrhoea and nausea. Meanwhile, a dangerous concerning complication is Stevens-Johnson syndrome, which can cause severe morbidity and mortality. Delayed hypersensitivity to allopurinol is rare but should be one of the differential diagnoses if a patient with underlying gout on chronic allopurinol treatment presents with skin rashes. The present case highlights the importance of a high index of suspicion in at-risk patients with underlying gout along with skin rashes on long-term allopurinol treatment to avoid unnecessary patient management.

Xanthine Oxidase Inhibitory Peptides from Larimichthys polyactis: Characterization and In Vitro/In Silico Evidence.

Hyperuricemia is linked to a variety of disorders that can have serious consequences for human health. Peptides that inhibit xanthine oxidase (XO) are expected to be a safe and effective functional ingredient for the treatment or relief of hyperuricemia. The goal of this study was to discover whether papain small yellow croaker hydrolysates (SYCHs) have potent xanthine oxidase inhibitory (XOI) activity. The results showed that compared to the XOI activity of SYCHs (IC50 = 33.40 ± 0.26 mg/mL), peptides with a molecular weight (MW) of less than 3 kDa (UF-3) after ultrafiltration (UF) had stronger XOI activity, which was reduced to IC50 = 25.87 ± 0.16 mg/mL (p < 0.05). Two peptides were identified from UF-3 using nano-high-performance liquid chromatography-tandem mass spectrometry. These two peptides were chemically synthesized and tested for XOI activity in vitro. Trp-Asp-Asp-Met-Glu-Lys-Ile-Trp (WDDMEKIW) (p < 0.05) had the stronger XOI activity (IC50 = 3.16 ± 0.03 mM). The XOI activity IC50 of the other peptide, Ala-Pro-Pro-Glu-Arg-Lys-Tyr-Ser-Val-Trp (APPERKYSVW), was 5.86 ± 0.02 mM. According to amino acid sequence results, the peptides contained at least 50% hydrophobic amino acids, which might be responsible for reducing xanthine oxidase (XO) catalytic activity. Furthermore, the inhibition of the peptides (WDDMEKIW and APPERKYSVW) against XO may depend on their binding to the XO active site. According to molecular docking, certain peptides made from small yellow croaker proteins were able to bind to the XO active site through hydrogen bonds and hydrophobic interactions. The results of this work illuminate SYCHs as a promising functional candidate for the prevention of hyperuricemia.

Chalcone derivatives as xanthine oxidase inhibitors: synthesis, binding mode investigation, biological evaluation, and ADMET prediction.

Xanthine oxidase (XO) is a crucial target for the treatment of hyperuricemia and gout. A series of derivatives based on natural 3,4-dihydroxychalcone, obtained from Carthamus tinctorious and Licorice, were designed and synthesized. Nine derivatives (9a-e, 10b,c, and 15a,b) exhibited apparent XO inhibitory activity in vitro (IC50 values varied from 0.121 to 7.086 µM), 15b presented the most potent inhibitory activity (IC50 = 0.121 µM), which was 27.47-fold higher than that of allopurinol (IC50 = 3.324 µM). The SAR analysis indicated that introducing hydroxyl groups at 3'/4'/5'-position on ring A was more beneficial to the inhibition of XO than at 2'/6'-position; the removal of 3­hydroxyl group on ring B could weaken the inhibitory potency of hydroxychalcones on XO, but it was beneficial to the XO inhibitory potency of methoxychalcones. Molecule modeling studies afforded insights into the binding mode of 15b with XO and supported the findings of SAR analysis. Additionally, kinetics studies demonstrated that 15b presented a reversible and competitive XO inhibitor, which spontaneously combined with XO through hydrophobic force, and finally changed the secondary conformation of XO. Furthermore, the acute hyperuricemia model was employed to investigate the hypouricemic effect of 15b, which could effectively reduce the serum uric acid levels of rats at an oral dose of 10 mg/kg. ADMET prediction suggested that compound 15b possessed good pharmacokinetic properties. Briefly, compound 15b emerges as an interesting XO inhibitor for the treatment of hyperuricemia and gout with beneficial effects on serum uric acid levels regulating. Meanwhile, the XO inhibitors with chalcone skeleton will deserve further attention and discussion.

Shikonin derivatives as potent xanthine oxidase inhibitors: in-vitro study.

Induction of hypersensitivity reactions (may be fatal too) by specific XO inhibitors has led to development of new molecules that are efficacious and have safer ADME profile. Among natural compounds, biologically active Alkannin/Shikonin (A/S) derivatives have unexplored XO inhibition potential. Therefore, their iso-hexenylnaphthazarin nucleus was studied and found that the nucleus is similar to that of allopurinol, signifying the XO inhibitory potential of these derivatives. For confirmation of their potential, ß,ß-dimethylacrylshikonin and deoxyshikonin were successfully isolated and characterised from Arnebia euchroma (Royle.) Johnst. (Boraginaceae) and were evaluated for in vitro XO inhibitory potential. ß,ß-dimethylacrylshikonin and deoxyshikonin showed a good XO inhibition potential with IC50 values of 7.475 ± 1.46 µg/mL and 4.487 ± 0.88 µg/mL, respectively. Results also validated the pharmacophore hypothesis, and it was concluded that nucleus iso-hexenylnaphthazarin can be remodelled for optimising the efficacy.

Therapeutic effects and mechanisms of N-(9,10-anthraquinone-2-ylcarbonyl) xanthine oxidase inhibitors on hyperuricemia.

Objective: To observe the antioxidative effects of N-(9,10-anthraquinone-2-ylcarbonyl) xanthine oxidase inhibitors (NAY) in vitro and in vivo models of hyperuricemia and explore the mechanism. Methods: A classical experimental method of acute toxicity and a chronic toxicity test were used to compare the toxic effects of different doses of NAY in mice. The hyperuricemia mouse model was established by gavage of potassium oxonate in vivo. After treatment with different doses of NAY (low dose: 10 mg/kg, medium dose: 20 mg/kg, and high dose: 40 mg/kg) and allopurinol (positive drug, 10 mg/kg), observe the levels of uric acid (UA), creatinine (CRE), and urea nitrogen (BUN) in urine and serum, respectively, and detect the activities of xanthine oxidase in the liver. The hyperuricemia cell model was induced by adenosine and xanthine oxidase in vitro. The cells were given different doses of NAY (50, 100, and 200 µmol/L) and allopurinol (100 µmol/L). Then the culture supernatant UA level of the medium was measured. The next step was to detect the xanthine oxidase activity in the liver and AML12 cells, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammatory factors in the kidney and serum of mice. Western blot was used to detect xanthine oxidase protein expression in mouse liver tissue and AML12 cells, ASC, Caspase-1, NLRP3, GLUT9, OAT1, and OAT3 protein expression in mouse kidney tissue and HK-2 cells. Hematoxylin-eosin staining was used to stain the liver and kidney tissues of mice and observe the tissue lesions. Results: NAY had little effect on blood routine and biochemical indexes of mice, but significantly reduced the serum UA level. NAY significantly reduced the level of UA in hyperuricemia mice and cells by inhibiting xanthine oxidase activity and reduced the levels of TNF-α, IL-6, and other inflammatory factors in serum and kidney of mice. NAY can inhibit inflammation by inhibiting the NLRP3 pathway. In addition, NAY can downregulate GLUT9 protein expression and upregulate OAT1 and OAT3 protein expression to reduce the UA level by promoting UA excretion and inhibiting UA reabsorption. Conclusion: These findings suggested that NAY produced dual hypouricemic actions. On the one hand, it can inhibit the formation of UA by inhibiting xanthine oxidase inhibitors activity, and on the other hand, it can promote the excretion of UA by regulating the UA transporter. It provides new ideas for the development of hyperuricemia drugs in the future.

A possible covalent xanthine oxidase inhibitor TS10: Inhibition mechanism, metabolites identification and PDPK assessment.

Xanthine oxidase (XO) inhibitors are widely used in the control of serum uric acid levels in the clinical management of gout. Our continuous efforts in searching novel amide-based XO inhibitors culminated in the identification of N-(4-((3-cyanobenzyl)oxy)-3-(1H-tetrazol-1-yl)phenyl)isonicotinamide (TS10), which exhibited comparable in vitro inhibition to that of topiroxostat (TS10, IC50 = 0.031 µM; topiroxostat, IC50 = 0.020 µM). According to the molecular modeling, we speculated that, as well as topiroxostat, TS10 would be biotransformed by XO to yield TS10-2-OH. In this work, TS10-2-OH was successfully identified in XO targeted metabolism study, demonstrated that TS10 underwent a covalent binding with XO via a TS10-O-Mo intermediate after anchoring in the XO molybdenum cofactor pocket. Furthermore, TS10-2-OH is a weak active metabolite, and its potency was explained by the molecular docking. In metabolites identification, TS10 could be oxidized by CYP2C9, CYP3A4 and CYP3A5 to generate two mono-hydroxylated metabolites (not TS10-2-OH); and could occur degradation in plasma to mainly generate a hydrolytic metabolite (TS10-hydrolysate). In pharmacokinetic assessment, the low oral system exposure was observed (Cmax = 14.73 ± 2.66 ng/mL and AUClast = 9.17 ± 1.42 h⋅ng/mL), which could be explained by the poor oral absorption property found in excretion studies. Nonetheless, in pharmacodynamic evaluation, TS10 exhibited significant uric acid-lowering effect after oral administration in a dose-dependent manner. Briefly, in addition to allopurinol and topiroxostat, TS10 is possibly another explicitly mechanism-based XO inhibitor with powerful covalent inhibition.